The central focus of the proposed studies is to delineate molecular mechanisms of eNOS uncoupling and endothelial dysfunction in type 2 diabetes mellitus (T2DM), and its contribution to hypertension, inflammatory activation and accelerated atherogenesis. During the previous funding cycle we have identified an angiotensin II (Ang II)-dependent mechanism that involves NOX1 (NADPH oxidase isoform 1) activation and NOX1-mediated uncoupling of eNOS and endothelial dysfunction. In preliminary experiments we have discovered a unique BMP4 (bone morphogenic protein 4)-dependent pathway that is differentially responsible for eNOS uncoupling and endothelial dysfunction in db/db T2DM. Further dissection of the BMP4-dependent mechanisms will ultimately promote development of novel therapies selectively targeting endothelial dysfunction in patients with T2DM. In Aim 1 we will determine whether and how eNOS uncoupling occurs in T2DM. In preliminary experiments circulating levels of BMP4, but not Ang II, was markedly elevated in T2DM mice. The second model of diet induced had time- dependent increase in body weight and a rapid response in increasing circulating BMP4 levels 2 weeks after high fat feeding. We will first determine circulating levels of BMP4 and its sources of production in T2DM; and then assess eNOS uncoupling activity with or without infusion of BMP4 antagonist noggin or in vivo RNAi of BMP4. Potential intermediate roles of NOX isoforms and eNOS cofactor enzyme deficiency in BMP4 uncoupling of eNOS and its consequences will also be evaluated (Aim 2), using NOXs/db/db double knockouts and eNOS cofactor enzyme transgenic/knockouts. Aim 2: To uncover pathophysiological consequences of BMP4 uncoupling of eNOS in T2DM. Blood pressure and endothelial function of aortas and mesenteric arteries will be assessed using telemetry and organbath/myobath in noggin infused hypertensive db/db mice. The inflammatory protein expression will also be examined in BMP4 stimulated aortic endothelial cells. In db/db/apoE null cross, noggin infusion or folic acid diet (known to recouple eNOS) will be tested for their efficacies in impeding atherogenesis. Aim 3: To determine how BMP4 activates NOX1 in T2DM which could lead to novel therapeutics targeting uncoupled eNOS to prevent endothelial dysfunction and atherogenesis. Our previous studies have established efficacy of in vivo RNAi for NOXO1, which will be utilized. Aortic endothelial cells will also be transfected of NOXO1 or p47phox prior to stimulation with BMP4 and analysis of NOXO1/p47phox-dependent NOX1 activity at different time points. By yeast 2-hybrid and co-IP experiments, a physical interaction between TLR2 and NOX1 has been established. Our preliminary findings demonstrated time-dependent increase in TLR2/NOX1 binding in response to BMP4 stimulation in aortic endothelial cells. RNAi and TLR2 null/db/db cross will be used to examine a role of TLR2 in BMP4- mediated eNOS uncoupling, endothelial dysfunction, hypertension and accelerated atherosclerosis in db/db mice. Overall the proposed studies will reveal important and innovative molecular mechanisms underlying development of vascular complications in T2DM, which would promote development of new therapeutics.